Assignment of functional amino acids around the active site of human DNA topoisomerase IIα

Y. Okada, Y. Ito, A. Kikuchi, Y. Nimura, S. Yoshida, M. Suzuki

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr805, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu794, Asp797, Ala801, and Arg804. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser803 and Lys798, which confer drug resistance, lie adjacent to this plane.

Original languageEnglish
Pages (from-to)24630-24638
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number32
DOIs
Publication statusPublished - 11-08-2000
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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